Peptoid and nonpeptoid containing alpha-keto oxadiazoles as serine protease inhibitors

ABSTRACT

The present invention relates to certain substituted oxadiazole peptoids and nonpeptoids useful as inhibitors of serine proteases, especially human neutophil elastase (HNE). Compounds of the present invention are useful for the treatment or amelioration of symptoms of adult respiratory distress syndrome, septic shock, and multiple organ failure. Processes mediated by HNE are also implicated in conditions such as arthritis, periodontal disease, glomerulonephritis, and cystic fibrosis.

This application is a continuation in part of U.S. Ser. No. 09/090,046,filed Jun. 3, 1998, now U.S. Pat. No. 6,001,813, and is a continuationin part of U.S. Ser. No. 09/090,274, filed Jun. 3, 1998, now U.S. Pat.No. 6,001,814; both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The serine proteases are a class of enzymes, which includes elastase,chymotrypsin, cathepsin G, trypsin and thrombin. These proteases have incommon a catalytic triad consisting of Serine-195, Histidine-57 andAspartic acid-102(chymotrypsin numbering system). Human neutrophilelastase (HNE) is a proteolytic enzyme secreted by polymorphonuclearleukocytes (PMNs) in response to a variety of inflammatory stimuli. Thisrelease of HNE and its extracellular proteolytic activity are highlyregulated and are normal, beneficial functions of PMNs. The degradativecapacity of HNE, under normal circumstances, is modulated by relativelyhigh plasma concentrations of α₁-proteinase inhibitor (α-PI). However,stimulated PMNs produce a burst of active oxygen metabolites, some ofwhich (hypochlorous acid for example) are capable of oxidizing acritical methionine residue in α-PI. Oxidized α-PI has been shown tohave limited potency as an HNE inhibitor and it has been proposed thatalteration of this protease/antiprotease balance permits HNE to performits degradative functions in localized and controlled environments.

Despite this balance of protease/antiprotease activity, there areseveral human disease states in which a breakdown of this controlmechanism is implicated in the pathogenesis of the condition. Impropermodulation of HNE activity has been suggested as a contributing factorin adult respiratory distress syndrome, septic shock and multiple organfailure. A series of studies also have indicated the involvement of PMNsand neutrophil elastase in myocardial ischemia-reperfusion injury.Humans with below-normal levels of α₁-PI have an increased probabilityof developing emphysema. HNE-mediated processes are implicated in otherconditions such as arthritis, periodontal disease, glomerulonephritis,dermatitis, psoriasis, cystic fibrosis, chronic bronchitis,atherosclerosis, Alzheimer's disease, organ transplantation, cornealulcers, and invasion behavior of malignant tumors.

There is a need for effective inhibitors of HNE as therapeutic and asprophylactic agents for the treatment and/or prevention ofelastase-mediated problems.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides compounds of formula I

wherein X and Y are independently O or N;

R₁ is alkyl α,α-dialkylalkylaryl or α,α-dialkylalkyl fusedaryl-cycloalkyl wherein the cycloalkyl group is optionally substitutedwith two or more O atoms;

R₂ and R₃ are independently H or alkyl; or together form a ringconsisting of 3-5 carbons in which one or more carbon atoms of the ringcan optionally be replaced with heteroatoms selected from O, S or Nwherein N is optionally substituted with H or alkyl, preferably one ofR₂ and R₃ is H and the other is iso-propyl; and

R₄ is alkyloxycarbonyl.

Preferably, compounds of the present invention comprise a 1,3,4oxadiazole ring (i.e., X is N; Y is O).

In one preferred embodiment of the invention, R₁ is alkyl, such astert-butyl. In another embodiment, R₁ is α,α-dialkylalkylaryl, such asan α,α-dimethylbenzyl group. In still another preferred embodiment, R₁is α,α-dialkylalkyl fused aryl-cycloalkyl wherein the cycloalkyl groupis substituted with two O atoms, such as anα,α-dimethyl-(3,4-methylenedioxy)benzyl group. In yet another preferredembodiment, R₂ and R₃ are independently alkyl, such as isopropyl, or H.Preferably, R₂ is isopropyl and R₃ is H.

In another embodiment, the present invention provides compounds offormula II:

wherein X and Y are independently O or N;

R₁, R₂, and R₃ are as above;

R′₂ and R′₃ are independently H or alkyl; or together form a ringconsisting of 3-5 carbon atoms in which one or more carbon atoms of thering can optionally be replaced by heteroatoms selected from O, S or N,wherein N is optionally substituted with H or alkyl;

A is a direct bond, —NH— or —OC(O)—NH—;

R₄ is H or halo; and

R₅ is H, alkyl or arylalkyl; or

a pharmaceutically acceptable salt thereof.

Preferably, compounds of this embodiment of the present inventioncomprise a 1,3,4 oxadiazole ring (i.e., X is N; Y is O).

In one preferred embodiment of the invention, R₁ is alkyl, such astert-butyl. In another embodiment, R₁ is α,α-dialkylalkyl fusedaryl-cycloalkyl wherein the cycloalkyl group is substituted with two Oatoms, such as an α,α-dimethyl-(3,4-methylenedioxy)benzyl group. In yetanother embodiment, R₁ is α,α-dialkylalkylaryl, such as anα,α-dimethylbenzyl group. In still another preferred embodiment, R₂ andR₃ are independently alkyl, such as isopropyl, or H. In a more preferredembodiment, R₂ is isopropyl, R₃ is H, and R₂′ and R₃′ are both H. WhereR₄ is halo, R₄ may be Cl, F, I or Br, although preferably it is F.

As used herein, the term “optionally substituted” means, whensubstituted, mono to fully substituted.

As used herein, the term “independently” means that the substituents maybe the same or different.

As used herein, the term “alkyl” means C₁-C₁₅, and preferably C₁-C₈. Itwill be understood that the alkyl group may be linear or branched.

As used herein, the term “α,α-dialkylalkylaryl” means that the alkylgroups are substituted at the α-positions to the oxadiazole ring or tothe aryl group or both. One such example is an α,α-dialkylbenzyl,wherein the α-substituents are preferably methyl, ethyl or propyl. Aspecific example is α,α-dimethylbenzyl. The term “α,α-dialkylalkyl fusedarylcycloalkyl” is defined to mean that the alkyl groups are substitutedat the α-positions to the oxadiazole ring or to the aryl group, and acycloalkyl is fused to the aryl ring. One such example of an“α,α-dialkylalkyl fused aryl-cycloalkyl” is anα,α-dialkyl-3,4-methylenedioxybenzyl group, wherein the α-substituentsare preferably methyl, ethyl or propyl; preferably they are methyl. Aspecific example includes the α, α-dimethyl-3,4-methylenedioxybenzylgroup.

As used herein, the term alkyloxycarbonyl means alkyl—O—C(O)— whereinthe meaning of alkyl is defined above. One such example of analkyloxycarbonyl is methyloxycarbonyl and is defined by the formulaCH₃—O—C(O)—.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the synthetic scheme for the Bocprotected amino alcohol intermediates used in the invention.

FIG. 2 is a schematic representation of the synthetic scheme for thecompounds of one embodiment of the invention.

FIG. 3 is a schematic representation of the synthetic scheme for thecompounds of another embodiment of the invention.

DETAILED DESCRIPTION

The compounds of the present invention have been found to be potentinhibitors of the serine protease human neutrophil elastase (HNE). Theyare reversible inhibitors that presumably form a transition stateintermediate with the active site serine residue. The compounds arecharacterized by their low molecular weights, high selectivity withrespect to HNE and stability regarding physiological conditions.Therefore, the compounds can be implemented to prevent, alleviate and/orotherwise treat diseases, which are mediated by the degradative effectsassociated with the presence of HNE. Their usage is of particularimportance as they relate to various human treatment in vivo but mayalso be used as a diagnostic tool in vitro.

The present invention provides, but is not limited to, specificembodiments set forth in the Examples as well as those set forth below.

The nomenclature for the embodiments is as follows (although embodimentsdisclosed indicate the stereochemistry of the 2-methylpropyl group ashaving the (S)-configuration, it will be understood that both theenantiomerically pure (R) and racemic (R,S) configurations are withinthe scope of the invention):

EXAMPLE 1Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.EXAMPLE 2Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.EXAMPLE 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.EXAMPLE 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[P-(2-[5-tert-butyl1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide. EXAMPLE 52-[5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.EXAMPLE 62-[5-Amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 72-[5-Benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.EXAMPLE 82-[5-Amino-6-oxo-2-phenyl-1,6dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 92-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 102-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 112-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-l-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 122-[6-Oxo-2-phenyl-1,6-dihydro4-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.EXAMPLE 132-[5-Methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(tert-butyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide.

The compounds of the present invention are not limited to use forinhibition of human elastase. Elastase is a member of the class ofenzymes known as serine proteases. This enzyme class also includes, forexample, chymotrypsin, cathepsin G, trypsin and thrombin. Theseproteases have in common a catalytic triad consisting of Serine-195,Histidine-57 and Aspartic acid-102 (chymotrypsin numbering system). Theprecise hydrogen bond network that exists between these amino acidresidues allows the Serine-195 hydroxyl to form a tetrahedralintermediate with the carbonyl of an amide substrate. The decompositionof this intermediate results in the release of a free amine and theacylated enzyme. In a subsequent step, this newly formed ester ishydrolyzed to give the native enzyme and the carboxylic acid. It is thiscarboxyl component that helps characterize the specificity for theenzyme. In the example in which the carboxyl component is a peptide, thealpha-substituent of the amino acid is predominately responsible for thespecificity toward the enzyme. Utilizing the accepted nomenclature bySchechter and Berger (Biochem. Biophy. Res. Commun., 27:157 (1967) andBiochem. Biophys. Res. Commun., 32:898 (1968)), the amino acid residuesin the substrate that undergo the cleavage are defined as P₁ . . . P_(n)toward the N-terminus and P₁′ . . . P_(n)′ toward the C-terminus.Therefore, the scissile bond is between the P₁ and the P₁′ residue ofthe peptide subunits. A similar nomenclature is utilized for the aminoacid residues of the enzyme that make up the binding pocketsaccormmodating the subunits of the substrate, where the binding pocketfor the enzyme is designated by S₁ . . . S_(n) instead of P₁ . . . P_(n)as for the substrate.

The characteristics for the P₁ residue defining serine proteinasespecificity is well established. The proteinases may be segregated intothree subclasses: elastases, chymases and tryptases based on thesedifferences in the P₁ residues. The elastases prefer small aliphaticmoieties such as valine whereas the chymases and tryptases prefer largearomatic hydrophobic and positively charged residues respectively.

One additional proteinase that does not fall into one of thesecategories is propyl endopeptidase. The P₁ residue defining thespecificity is a proline. This enzyme has been implicated in theprogression of memory loss in Alzheimer's patients. Inhibitorsconsisting of α-keto heterocycles have recently been shown to inhibitpropyl endopeptidase (Tsutsumi et al., J. Med. Chem., 37, 3492-3502(1994)). By way of extension, α-keto heterocycles as defined hereinallow for an increased binding in P′ region of the enzyme.

TABLE 1 P₁ Characteristics for Proteinase Specificity Proteinase ClassRepresentative Enzyme P₁ Characteristic Elastases Human NeutrophilElastase small aliphatic residues Chymases alpha-Chymotrypsin, aromaticor large Cathepsin G hydrophobic residues Tryptases Thrombin, Trypsin,positively charged Urokinase, Plasma residues Kallikrein, PlasminogenActivator, Plasmin Other Prolyl Endopeptidase proline

Since the P₁ residue predominately defmes the specificity of thesubstrate, the present invention relates to P₁-P_(n)′ modifications,specifically, certain alpha-substituted keto-heterocycles composed of1,2,4 oxadiazoles and 1,3,4-oxadiazoles. By altering thealpha-substituent to the ketone and, to some extent, the substituent onthe heterocycle, the specificity of these compounds can be directedtoward the desired proteinase (e.g., small aliphatic groups forelastase).

The efficacy of the compounds for the treatment of various diseases canbe determined by scientific methods, which are known in the art. Thefollowing are noted as examples for HNE mediated conditions:

for acute respiratory distress syndrome, the method according to humanneutrophil elastase (HNE) model (AARD, 141:227-677 (1990)); theendotoxin induced acute lung injury model in minipigs (AARD, 142:782-788(1990)); or the method according to human polymorphonuclearelastase-induced lung hemorrhage model in hamsters (European PatentPublication No. 0769498) may be used;

in ischemia/reperfusion, the method according to the canine model ofreperfusion injury (J. Clin. Invest., 81: 624-629 (1988)) may be used.The compounds of the present invention, salts thereof, and theirintermediates can be prepared or manufactured as described herein or byvarious processes known to be present in the chemical art (see e.g., WO96/16080).

Alternatively, the compounds of the present invention may be prepared asdescribed in FIGS. 1, 2 and 3. FIG. 1 relates to the synthesis of theBoc protected amino alcohol intermediates used in the invention. FIGS. 2and 3 show the use of the intermediates for the synthesis compounds ofthe invention.

The 2-substituted 1,3,4-oxadiazoles (3) may be prepared via formation ofmethyl esters from the corresponding acids (1) utilizing, for example,thionyl chloride and methanol, followed by treatment with hydrazine in asuitable solvent to yield hydrazonic acids (2). Alternatively, esterscan be prepared by methods known to one skilled in the art or thosemethods described in Comprehensive Organic Transformations (R. Larock,VCH Publishers 1989, 966-972). Reaction of (2) with triethylorthoformate or trimethyl orthoformate and TsOH gives the requisite2-substituted 1,3,4-oxadiazoles (3).

Intermediate (3′) can be formed utilizing standard conditions (e.g.,butyllithium. or other known alkyl lithium reagents, at low temperaturein a polar aprotic solvent, and further, if desired, reacting withMgBr·OEt₂) and subsequently added to aldehyde (4) to give alcohol (5).

The aldehyde (4) may be prepared via any of three methods as describedin FIG. 1. One method reduces the intermediate that is formed betweenBoc-Val-OH and iso-propylchloroformate with sodium borohydride to giveBoc-Valinol (12). In a subsequent step, the Boc-Valinol is oxidized withSO₃-Py in DMSO to give aldehyde (4). Another such method takes theWeinreb amide (13) that is prepared from Boc-Val-OH (11) and reduces itto the aldehyde using diisobutylaluminum hydride (DIBAL). Alternatively,one may generate the ester (14) of the amino acid followed by reductionwith DIBAL to afford aldehyde (4).

As shown in FIGS. 2 and 3, deprotection of amine (5) using hydrochloricacid in dioxane gives the amino hydrochloride (6), which is then coupledto the desired acid (7) or (7′) by methods available to one skilled inthe art to give intermediate (8) or (8′). Oxidation using the SwermOxidation, Dess-Martin's Periodinane or other methods as described inOxidation in Organic Chemistry (M. Hudlicky, ACS Monograph 186 (1990))yields the desired ketone (9) or (9′).

Where a compound is substituted at the 5 position of the pyrimidinonegroup with a benzyloxycarbonylamino group, a deprotection step can beconducted as described in FIG. 3. This step requires removal of theprotecting group from the amine and may be carried out by a number ofmethods. For example, one may utilize aluminum chloride, anisole andnitromethane in a suitable solvent such as dichloromethane to give the5-amino compound (10′). Other methods of deprotection available in theart may also be used.

Although the compounds described herein may be administered as purechemicals, it is preferable to present the active ingredient as apharmaceutical composition. The invention thus further provides the useof a pharmaceutical composition comprising one or more compoundstogether with one or more pharmaceutically acceptable carriers thereofand, optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be ‘acceptable’ in the sense of being compatible withthe other ingredients of the composition and not deleterious to therecipient thereof.

Pharmaceutical compositions include those suitable for oral orparenteral (including intramuscular, subcutaneous and intravenous)administration. The compositions may, where appropriate, be convenientlypresented in discrete unit dosage forms and may be prepared by any ofthe methods well known in the art of pharmacy. Such methods include thestep of bringing into association the active compound with liquidcarriers, solid matrices, semi-solid carriers, finely divided solidcarriers or combination thereof, and then, if necessary, shaping theproduct into the desired delivery system.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete unit dosage forms such as hard or soft gelatincapsules, cachets or tablets each containing a predetermined amount ofthe active ingredient; as a powder or as granules; as a solution, asuspension or as an emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art, e.g.,with enteric coatings.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspension, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, emulsifying agents, non-aqueousvehicles (which may include edible oils), or preservatives.

The compounds may also be formulated for parenteral administration(e.g., by injection, for example, bolus injection or continuousinfusion) and may be presented in unit dose form in ampules, pre-filledsyringes, small bolus infusion containers or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilization from solution, for constitution with a suitable vehicle,e.g., sterile, pyrogen-free water, before use.

For topical administration to the epidermis, the compounds may beformulated as ointments, creams or lotions, or as the active ingredientof a transdermal patch. Suitable transdermal delivery systems aredisclosed, for example, in Fisher et al. (U.S. Pat. No. 4,788,603) orBawas et al. (U.S. Pat. No. 4,931,279, 4,668,504 and 4,713,224).Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifing agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. The active ingredient can also be delivered viaiontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122,4,383,529, or 4,051,842.

Compositions suitable for topical administration in the mouth includeunit dosage forms such as lozenges comprising active ingredient in aflavored base, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert base such as gelatin andglycerin or sucrose and acacia; mucoadherent gels, and mouthwashescomprising the active ingredient in a suitable liquid carrier.

When desired, the above-described compositions can be adapted to providesustained release of the active ingredient employed, e.g., bycombination thereof with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

The pharmaceutical compositions according to the invention may alsocontain other adjuvants such as flavorings, coloring, antimicrobialagents, or preservatives.

It will be further appreciated that the amount of the compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular salt selected but also with the routeof administration, the nature of the condition being treated and the ageand condition of the patient and will be ultimately at the discretion ofthe attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg/day, e.g., from about 1 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form, forexample, containing 0.5 to 1000 mg, conveniently 5 to 750 mg, and mostconveniently, 10 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, more preferably, about 1 to 50 μM, and most preferably, about 2to about 30 μM. This may be achieved, for example, by the intravenousinjection of a 0.05 to 5% solution of the active ingredient, optionallyin saline, or orally administered as a bolus containing about 0.5-500 mgof the active ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may be conveniently presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations, such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

The following examples are given to illustrate the invention and are notintended to be inclusive in any manner.

EXAMPLES

The compounds of the present invention, salts thereof, and theirintermediates can be prepared or manufactured as described herein or byvarious processes known to be present in the chemical art. By way of anexample, the final step in the process defined here, is an oxidation ofa 2° alcohol to a ketone. As described here, this transformation from analcohol to ketone was preformed using dimethylsulfoxide and oxalylchloride followed by base, which is known as the Swern oxidation.However, modifications of the Swern oxidation are known in the art andare acceptable in this present invention. It is known that alternativeelectrophilic molecules can be substituted for oxalyl chloride such asdicyclohexylcarbodiimide, acetic anhydride, trifluoroacetic anhydride orsulfur trioxide (Mancuso et al., Synthesis 165 (1981)). Alternatively,other oxidative methods can be used such as N-chlorosuccinimide (NCS)followed by base as described by the inventors in U.S. Pat. No.5,618,792 or periodinane such as the Dess-Martin reagent. Still othermethods may also be appropriate as described in Oxidation in OrganicChemistry (M. Hudlicky, ACS Monograph 186 (1990)).

Besides the methods described below, other methods can be used formaking substituted oxadiazole nonpeptides. U.S. Pat. No. 5,807,829,incorporated herein by reference, teaches some other methods for makingsubstituted oxadiazole nonpeptides.

The skilled artisan will understand that where a particular enantiomeris mentioned, the mirror-image enantiomer or a mixture of enantiomerscan be used.

Symbols have the standard meanings as familiar to one skilled in theart, by way of example the following have been used: ml (milliliters), g(grams), TLC (thin layer chromatography), R_(f) (the ratio of thedistance moved by a compound to the distance that the solvent frontmoved during the same time on a TLC plate), ¹H NMR (proton nuclearmagnetic resonance), DMSO-d6 (deuterodimethylsulfoxide) and CDCl₃(deuterochloroform).

Example 1Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

The secondary alcohol,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide, was oxidized using oneof the methods known to one skilled in the art, such as, the SwemOxidation. The intermediatemethyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide was prepared asfollows:

A. tert-Butylcarbohydrazonic Acid

The mixture of methyl trimethylacetate (230 ml) and hydrazinemonohydrate (170 ml) was refluxed for 24 hours. The reaction was cooledto room temperature, and concentrated under reduced pressure. Theresidue was azeotroped with toluene several times, dissolved in asaturated aqueous solution of sodium chloride, and extracted withchloroform (4×). The extract was dried over anhydrous sodium sulfate,and concentrated under reduced pressure to givetert-butylcarbohydrazonic acid (176 g) having the following physicaldata.

TLC: R_(f)=0.59, chloroform: methanol (10:1).

¹H NMR (DMSO-d₆): δ 68.78 (1H, brs), 4.15 (2H, brs), 1.08 (9H, s).

B. 2-tert-Butyl-1,3,4-oxadiazole

The mixture consisting of tert-butylcarbohydrazonic acid (176 g),trimethyl orthoformate (250 ml) and p-toluenesulfonic acid monohydrate(4.3 g) was heated and methanol removed by distillation at a temperatureranging from 90° C. to 110° C. Trimethyl orthoformate was removed (50°C./43 mm Hg) and the residue was distilled at 120° C./23 mm Hg to give2-tert-Butyl-1,3,4-oxadiazole (131 g) having the following physicaldata.

TLC: R_(f)=0.68, chloroform: methanol (10:1).

¹H NMR (DMSO-d₆): δ 9.12(1H, s), 1.3 6 (9H, s).

C.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol

To a solution of 2-tert-Butyl-1,3,4-oxadiazole (62.1 g) intetrahydrofuran (1650 ml) was added n-butyllithium in hexane (1.6 M,307.8 ml) dropwise at −78° C. under an atmosphere of argon. The mixturewas stirred for 40 min at −78° C., magnesium bromide diethyl etherate(127.2 g) was added, and the resulting mixture was allowed to warm to−45° C. After 1.5 hours, a solution of2-(S)-[N-(tert-butoxycarbonyl)amino]-3-methylbutanal (90 g) intetrahydrofuran (60 ml) was added dropwise at -45° C. and allowed towarm to −15° C. The reaction mixture was quenched by addition of asaturated aqueous solution of ammonium chloride, and extracted withethyl acetate. The extract was washed with water (×3) and a saturatedaqueous solution of sodium chloride, dried over anhydrous sodium sulfateand concentrated. The residue was purified by column chromatography onsilica gel (Merck 7734) (ethyl acetate:hexane=1:20 to 1:1) to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(78.6 g) having the following physical data.

TLC: R_(f)=0.42, hexane:ethyl acetate (1:1).

¹H NMR (CDCl₃): δ 5.16-4.90 (2H, m), 4.67 (1H, m), 4.23 (1H, m), 3.90(1H, m), 3.66 (1H, m), 1.98 (1H, m), 1.42, 1.41 and 1.36 (total 18H,each s), 1.13-0.90 (6H, m).

D. 1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olHydrochloride

To a solution of1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(76.3 g) in dioxane (200 ml) was added 4N hydrochloric acid in dioxanesolution (1000 ml) at 0° C. The reaction mixture was concentrated underreduced pressure. The residue was solidified with diethyl ether. Thesolid was azeotroped with benzene several times to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride (66.1 g) having the following physical data.

TLC: R_(f)=0.30, chloroform:methanol (10:1);

¹H NMR (CDCl₃): δ 8.50-8.10 (2H, br), 7.10-6.80 (1H, br), 5.55-5.35 (1H,m), 3.95-3.60 (2H, m), 2.10 (1H, m), 1.41 (9H, s), 1.20-1.00 (6H, m).

E.Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide

Prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride and a coupling method known to one skilled in the art.

The product had the following physical data.

TLC: R_(f)=0.58, ethyl acetate.

¹H NMR: (200 MHz, CDCl₃), δ 7.53 (brd., J=6.2 Hz, 1H, NH), δ 5.45-5.29(m 2H, NH, and α CH of P₁ Val), δ 4.79-4.62 (m, 1H, α CH of Pro), 4.32(m, 1H, α CH of P₃-Val), 3.83-3.51 (m, 2H, NCH₂ of Pro), 3.68 (s, 3H,CH₃O), 2.55-1.80 (m, 6H, CHs of iso-Pr, and CH₂CH₂ of Pro), 1.47 (s, 9H,CH₃s of t-Bu), 1.16-0.86 (m, 12H, CH₃s of iso-Pr).

Example 2Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide

The compound was prepared by oxidizingmethyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamideusing a procedure known to one skilled in the art, such as, the SwernOxidation.

The intermediate,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethylbenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamide,was prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride and a coupling method know to one skilled in the art. Theintermediate1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 except methyl phenylisobutyrate was used instead of methyltrimethylacetate.

The product had the following physical data.

TLC: R_(f)=0.64, ethyl acetate.

¹H NMR (200 MHz, CDCl₃): 7.84 and 7.49 (each brd., J=7.6 Hz, totally 1H,NH), 7.40-7.20 (m, 5H aromatic Hs), 5.46-5.29 (m, 2H, NH and α CH of P₁Val), 4.77-4.60 (m, 1H, α CH of Pro), 4.40-4.25 (m, 1H α CH of P₃ Val),3.84-3.55 (m, 2H, NCH₂ of Pro), 3.68 (s, 3H, CH₃O), 2.55-1.76 (m, 6H,CHs of iso-Pr and CH₂CH₂ of Pro), 1.88 (s, 6H, hetC(CH₃)₂Ph), 1.12-0.82(m, 12H, CH₃s of iso-Pr).

Example 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylene-dioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide

The compound was prepared by oxidizingmethyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinamideusing a procedure know to one skilled in the art, such as, the SwernOxidation.

The intermediate,methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]-L-prolinade,was prepared using methyloxycarbonyl-L-Val-Pro-OH and1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-olhydrochloride and a coupling method know to one skilled in the art. Theintermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 except methyl 3,4-methylenedioxyphenylisobutyrate was usedinstead of methyl trimethylacetate.

The product had the following physical data.

TLC: R_(f)=0.63, ethyl acetate.

¹H NMR (200 MHz, CDCl₃): 7.49 (d, J=6.4 Hz, 1H, NH), 6.85-6.73 (m, 3H,aromatic Hs), 5.95 (s, 2H, OCH₂O), 5.46-5.28 (m, 1H α CH of Pro), 4.30(m, 1H, α CH of P₃-Val), 3.84-3.54 (m, 2H, NCH₂ of Pro), 3.68 (s, 3H,CH₃O), 2.55-1.78 Pr, and CH ₂CH₂ of Pro), 1.83 (s, 6H, HetC(CH₃)2Ph),1.11-0.85 (m, 12H, CH₃s of iso-Pr).

Example 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide

To a solution of oxalyl chloride (5.80 ml) in dichloromethane (160 ml)was slowly added dropwise a solution of dimethylsulfoxide (9.44 ml) indichloromethane (16 ml) at −78° C. under an atmosphere of argon. Themixture was stirred for 30 min at 78° C. To the mixture was addeddropwise a solution of2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamide(15.2g) in dichloromethane (160 ml) at −78° C. The mixture was stirredfor 2 hours at −78° C. To the resulting solution was added triethylamine(97.2 ml) dropwise at −78° C. The reaction mixture was warmed up to roomtemperature, and stirred for 34 hours at the same temperature. Thereaction mixture was acidified by addition of 2N aqueous solution ofhydrochloric acid, and extracted with dichloromethane. The extract waswashed with 2N aqueous solution of hydrochloric acid, water and asaturated aqueous solution of sodium chloride, dried over anhydroussodium sulfate, and concentrated. The residue was purified by columnchromatography on silica gel using a gradient elution of 66 to 100%ethyl acetate/hexane to give2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide(10.92 g) having the following physical data.

TLC: R_(f)=0.63, chloroform:methanol (10:1).

¹H NMR (CDCl₃): δ 8.00(1H, d, J=6.5 Hz), 7.64 (2H, dd, J=8.6, 5.4 Hz),7.17 (2H, t, J=8.6 Hz), 6.95 (IH, brd, J=8.4 Hz), 6.50 (IH, d, J=6.5Hz), 5.43 (IH, dd, J=8.4, 4.8 Hz), 4.63 and 4.58 (each 1H, each d,J=15.4 Hz), 2.53 (IH, m), 1.48 (9H, s), 1.09 (3H, d, J=6.8 Hz), 0.90(3H, d, J=6.8 Hz). The intermediate2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared as follows:

A. tert-Butylcarbohydrazonic acid t-Butylcarbohydrazonic acid wasprepared as described above. TLC: R_(f)=0.59, chloroform:methanol(10:1). ¹H NMR (DMSO-d₆): δ 8.78 (1 H, brs), 4.15 (2H, brs), 1.08 (9H,s).

B. 2-tert-Butyl 1,3,4-oxadiazole t-tert-Butyl 1,3,4-oxadizole wasprepared as described above.

C.1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol

To a solution of 2-tert-Butyl-1,3,4-oxadiazole (62.1 g) intetrahydrofuran (1650 ml) was added n-butyllithium in hexane (1.6 M,307.8 ml) dropwise at −78° C. under an atmosphere of argon. The mixturewas stirred for 40 min at −78° C., magnesium bromide diethyl etherate(127.2 g) was added, and the resulting mixture was allowed to warm to−45° C. After 1.5 hours, a solution of2-(S)-[N-(tert-butoxycarbonyl)amino]-3-methylbutanal (90 g) intetrahydrofuran (60 ml) was added dropwise at −45° C. and allowed towarm to −15° C. The reaction mixture was quenched by addition of asaturated aqueous solution of ammonium chloride, and extracted withethyl acetate. The extract was washed with water (×3) and a saturatedaqueous solution of sodium chloride, dried over anhydrous sodium sulfateand concentrated. The residue was purified by column chromatography onsilica gel (Merck 7734) (ethyl acetate:hexane=1:20→1:1) to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(78.6 g) having the following physical data.

TLC: R_(f)=0.42, hexane:ethyl acetate (1:1).

¹H NMR (CDCl₃): δ 5.16-4.90 (2H, m), 4.67 (1H, m), 4.23 (1H, m), 3.90(1H, m), 3.66 (1H, m), 1.98 (1H, m), 1.42, 1.41 and 1.36 (total 18H,each s), 1.13-0.90 (6H, m).

D. 1-[2-(5-tert-Butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olHydrochloride

To a solution of1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-(tert-butoxycarbonylamino)-3-methylbutan-1-ol(76.3 g) in dioxane (200 ml) was added 4N hydrochloric acid in dioxanesolution (1000 ml) at 0° C. The reaction mixture was concentrated underreduced pressure. The residue was solidified with diethyl ether. Thesolid was azeotroped with benzene several times to give1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-ainino-3-methylbutan-1-olhydrochloride (66.1 g) having the following physical data.

TLC: R_(f)=0.30, chloroform:methanol (10:1);

¹H NMR (CDCl₃): δ 8.50-8.10 (2H, br), 7.10-6.80 (1 H, br), 5.55-5.35(1H, m), 3.95-3.60 (2H, m), 2.10 (1H, m), 1.41 (9H, s), 1.20-1.00 (6H,m).

E.2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrinidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazol]hydroxymethyl-)2-(S)-methylpropyl]acetamide

To a solution of1-[2-(5-tert-butyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride (10.76 g),[6-oxo-2-(4flurophenyl)-1,6-dihydro-1-pyrimidinyl]acetic acid (8.63 g)and 1-hydroxybenzotriazole (5.85 g) in dimethylformamide (100 ml) wasadded 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide (7.33 g) at 0° C.To the resulting mixture was added 4-methylmorpholine (4.21 ml) at thesame temperature. The reaction mixture was stirred for 17 hours at roomtemperature. The reaction was quenched by addition of water, extractedwith ethyl acetate (×3). The extract was washed with aqueous 10% citricacid solution, a saturated aqueous solution of sodium hydrogencarbonate,water and a saturated aqueous solution of sodium chloride. The organicphase was dried over anhydrous sodium sulfate and concentrated underreduced pressure to2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrnmidinyl]-N-[1-(2-[5-tertbutyl1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetarnide (14.6 g)having the following physical data.

TLC: R_(f)=0.40, chloroform:methanol (10:1);

¹H NMR (DMSO-d₆): δ 8.00 and 7.94 (each 1H, each d, J=6.6 Hz), 7.71 and7.55 (each 2H, each m), 7.19 and 7.18 (each 2H, each J=6.6 Hz), 6.43 and6.3 8 (each 1H, each d, J=6.6 Hz), 5.13 (1H, d, J=2.2 Hz), 5.05 (1H, d,J=4.4 Hz), 4.54 (2H, s), 4.43 (2H, m), 4.31 (1H, m), 4.04 (1H, m),2.20-1.52 (1H, m), 1.41 and 1.37 (each 9H, each s), 1.08, 1.00, 0.94 and0.92 (each 3H, each d, J=6.6 Hz).

Example 52-[5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-l-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide

The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidefor the 2° alcohol. The title compound,2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide,gave the following physical data.

TLC: R_(f)=0.66, chloroformn:methanol (10:1);

¹H NMR (CDCl₃): δ 8.76 (1H, brs), 7.63-7.52 (2H, m), 7.49 (1H, brs),7.38 (5H, brs), 7.13 (2H, t, J=8.6 Hz), 6.82-6.74 (3H, m), 6.71 (1H,brd, J=8.6 Hz), 5.94 (2H, s), 5.42 (1H, dd, J=8.6, 5.0 Hz), 5.22 (2H,s), 4.58 (2H, brs), 2.50 (1H, m), 1.83 (6H, s), 1.05 and 0.86 (each 3H,each d, J=7.0 Hz).

The intermediate2-[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analoguous manner as described in Example 1 E using[5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]aceticacid and1-[2(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazole gave thefollowing physical data.

TLC: R_(f)=0.69, chloroform:methanol (10:1).

¹ ^(H NMR (CDCl) ₃): δ 8.30 (1H, s), 6.78(1H, brs), 6.74(2H, brs), 5.94(2H, s), 1.81 (6H, s).

Example 62-[5Amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

To2-[5-(benzyloxycarbonylamino)-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide(1.42 g) was added 30% hydrobromic acid in acetic acid solution (50 ml).The reaction mixture was stirred for 1 hour at room temperature. Thereaction mixture was quenched by addition of ice water, extracted withethyl acetate (×2). The combined extracts were washed with water (×2)and a saturated aqueous solution of sodium chloride. The organic phasewas dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure. The residue was purified by column chromatography onsilica gel using a gradient elution of 50 to 100% ethyl acetate/hexaneto give2-[5-amino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide(457 mg) having the following physical data.

TLC: R_(f)=0.39, ethyl acetate.

¹H NMR (CDCl₃): δ 7.53 (2H, dd, J=8.8, 5.3 Hz), 7.48 (1H, s), 7.06 (2H,t, J=8.8 Hz), 6.90 (1H, brd, J=8.4 Hz), 6.84-6.70 (3H, m), 5.95 (2H, s),5.43 (1H, dd, J=8.4, 4.8 Hz), 4.63 and 4.54 (each 1H Abq, J=15.0 Hz),4.05 ((2H, brs), 2.51 (1H, m), 1.84 (6H, s), 1.06 and 0.87 (each 3H,each d, J=7.0 Hz).

Example 72-[5-Benzyloxycarbonylamino-6-oxo-2-pheny-1-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro4-pyrimidinyl]-N-[1-(2-[-5(α,α-dimethyl-3,4methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyljacetamidefor the 2° alcohol. The title compound,2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acemidegave the following physical data.

TLC: R_(f)=0.34, hexane:ethyl acetate (1:1).

¹H NMR (CDCl₃): δ 8.78 1H, brs), 7.60-7.30 (1H, m), 6.78 (3H, m), 6.68(1H, d, J=8.8 Hz), 5.94 (2H, s), 5.42 (1H, dd, J=8.8, 4.8 Hz), 5.23 (2H,s), 4.65 and 4.57 (2H, Abq, J=15.0 Hz), 2.49 (IH, m), 1.83 (6H, s), 1.04(3H, d, J=6.0 Hz), 0.84 (3H, d, J=5.8 Hz).

The intermediate2-[5-benzyloxycarbonylamino-6-oxo-2phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analogous manner as described in Example 1 E using5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]aceticacid and1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazole gave thefollowing physical data.

TLC: R_(f)=0.69, chloroform: methanol (10:1).

¹H NMR (CDCl₃): δ 8.30 (1H, s), 6.78(1H, brs), 6.74(2H, brs), 5.94 (2H,s), 1.81 (6H, s).

Example 82-[5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

The compound was prepared using a similar procedure as described inExample 3 utilizing2-[5-benzyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,αdimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide.The title compound2-[5-Amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamidegave the following physical data.

TLC: R_(f)=0.40, ethyl acetate.

¹H NMR (CDCl₃): δ 7.59-7.34 (5H, m), 7.50 (1H, s), 6.86 (1H, d, J=8.2Hz), 6.86-6.72 (3H, m), 5.95 (2H, s), 5.43 (1H, dd, J=8.2 and 4.8 Hz),4.66 1H, d, J=15.4 Hz), 4.56 (2H, f, J=15.4 hz), 4.05 (2h, brs),2.62-2.36 (1H, m), 1.84 (6H, s), 1.05(3H, d, J=7.0 Hz), 0.85 (3H, d,J=7.0 Hz).

Example 92-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

TLC: R_(f)=0.46, ethyl acetate.

¹H NMR (CDCl₃): δ 8.01 (1H, d, J=6.6 Hz), 7.65-7.35 (5H, m), 6.87 (1H,d, J=8.6,Hz), 6.85-6.70 (3H, m), 6.49 (1H, d, J=6.6 Hz), 5.95 (2H, s),5.42 (1H, dd, J=8.6 and 5.0 Hz), 4.67 (1H, d, J=15.2 Hz), 4.54 (1H, d,J=15.2 Hz), 2.63-2.37 (1H, m), 1.84 (6H, s), 1.05 (3H, d, J=6.8 Hz),0.85 (3H, d, J=6.8 Hz)

Example 102-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

TLC: R_(f)=0.43, ethyl acetate.

¹H NMR (CDCl₃): δ 7.99 (1H, d, J=6.6 Hz), 7.63 (2h, dd, J=8.6, 5.2 Hz),7.14 (2H, t, J=8.6 Hz), 6.93 (1H, brd, J=8.6 Hz), 6.84-6.70 (3H, m),6.49 (1H, d, J=6.6 Hz), 5.95 (2H, s), 5.41 (1H, dd, J=8.6, 5.0 Hz), 4.64and 4.53 (each 1H, Abq, J=I 5.0 Hz), 2.50 (1H, m), 1.84 (6H, s), 1.06and 0.87 (each 3H, each d, J=7.0 Hz).

Example 112-[6-oxo-2-(4-fluorophenyl-)1,6-dihydro4-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)methylpropyl]acetamide

The compound was prepared using a similar oxidative procedure asdescribed in Example 1 utilizing2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidefor the 2° alcohol. The title compound,2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenryl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]acetamide,gave the following physical data.

TLC: R_(f)=0.42, ethyl acetate.

¹H NMR (CDC 13): δ 7.99 (1H, d, J=6.5 Hz), 7.62 (2H, m), 7.40-7.20 (5H,m), 7.14 (2H, t, J=8.8 Hz), 6.89 (1H, brd, J=8.6 Hz), 6.49 (1H, d, J=6.5Hz), 5.42 (1H, dd, J=8.6, 5.0 Hz), 4.61 and 4.54 (each 1H, each d,J=15.0 Hz), 2.50 (1H, m), 1.88 (6H, s), 1.06 and 0.86 (each 3H, each d,J=6.71 Hz).

The intermediate2-[6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analoguous manner as described in Example 1 E using[6-oxo-2-(4-fluoropheny-1)1,6-dihydro-1-pyrimidinyl]acetic acid and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride. The intermediate1-[2-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrocloride was prepared using a similar procedure as described inExample 1 D. The heterocyclic intermediate2-(α,α-dimethylbenyl)-1,3,4-oxadiazole gave the following physical data.

TLC: R_(f)=0.43, ethyl acetate:hexane (1:2).

¹H NMR (CDCl₃): δ 8.31 (1H, s), 7.40-7.14 (5H, m), 1.86 (6H, s).

Example 122-[6-oxo-2-phenyl-1,6-dihydro-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

The compound was prepared using a similar oxidative procedure asdescribed in Example 4 utilizing2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidefor the 2° alcohol. The title compound,2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(S)-methylpropyl]atetamide,gave the following physical data.

TLC: R_(f)=0.44, ethyl acetate.

¹H NMR (CDCl₃): δ 8.02 (1H, d, J=6.5 Hz), 7.64-7.24 (10H, m), 6.82 (1H,brd, J=8.4 Hz), 6.50 (1H, d, J=6.5 Hz), 5.44 (1H, dd, J=8.4, 4.8 Hz),4.63 and 4.56 (each 1H, each d, J=15.4 Hz), 2.50 (1H, m), 1.89 (6H, s),1.06 and 0.86 (each 3H, each d, J=6.8 Hz).

The intermediate2-[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]hydroxymethyl)-2-(S)-methylpropyl]acetamidewas prepared in an analoguous manner as described in Example 1 E using[6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]acetic acid and1-[2-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]-2-(S)-amino-3-methylbutan-1-olhydrochloride.

Example 132-[5-Methyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro4-pyrirnidinyl]-N-[1-(2-[5-(tert-butyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)methylpropyl]acetamide.

Prepared by a procedure analogous to that of Example 7. The product hadthe following physical data.

TLC: R_(f)=0.57methanol:chloroform, 1:10.

¹H NMR (200 MHz, CDCl₃): 8.78 (brs, 1H, H of pyrimidinone), 7.62-7.40(m, 6H, NH and aromatic Hs), 6.73 (brd, J=8.4 Hz, 1H, CONH), 5.45 (dd,J=8.4, 5.0 Hz, 1H, α CH of Val), 4.67 and 4.61 (each d, J=15.0 Hz, each1H, CH₂ of Gly), 3.81 (s, 3H, CH₃O), 2.512 (m, 1H, CH of iso-Pr), 1.48(s, 9H, CH₃s of t-Bu), 1.07 and 0.88 (each d, J=6.8 Hz, each 3H, CH3s ofiso-Pr).

Example 14 In Vitro Inhibition of Elastase

The following protocol was used to determine inhibitory activity ofcompounds described herein. The elastase used in the protocol wasderived from human sputum (HSE). A mother solution of the HSE enzyme wasprepared from commercially available HSE (875 U/mg protein, SE-563,Elastin Product Co., Inc, Missouri, USA) by diluting with saline to1,000 U/ml, which was flirdier diluted to 2 U/ml at 0° C. prior to use.

A solution was prepared by mixing 100 μl 0.2 M HEPES-NaOH buffer (pH8.0), 40 μl 2.5 M NaCl, 20 μl 1% polyethyleneglycol 6000, 8 μl distilledwater, 10 μl of a DMSO solution of inhibitor and 2 μl solution ofN-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (SEQ ID NO:1)(atconcentrations of 100, 200 and 400 μM). The solution was incubated for10 minutes at 37° C. To this was added an enzyme solution of HSE(elastase derived from human sputum). The resulting mixture wassubjected to the following rate assay.

Optical density (SPECTRA MAX 250, Molecular Devices) at 405 nm due top-nitroaniline generated by the enzyme reaction was measured at 37° C.in order to measure the reaction rate during the period that theproduction rate of p-nitroaniline remains linear. The rate, mO.D./min.,was measured for 10 minutes at 30 second intervals immediately after theaddition of the enzyme solution. IC₅₀ values were determined bylog-logit method and converted to K_(I) values by Dixson plot method.The compounds are presented in Table 2 g the inhibition activity (K_(I)values, nM) against HNE.

TABLE 2 Biological Activity Example Name K_(I) (nM) 1Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(tert-butyl)-  3.01,3,4-oxadiazolyl]carbonyl)-2-(S)-methyl-propyl]- L-prolinamide 2Methyloxycarbonyl-L-valyl-N-[1-(2-[5- 1.32(α,αdimethylbenzyl)oxadiazolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide. 3Methyloxycarbonyl-L-valyl-N-[1-(2-[5-(α,α-di- 0.24methyl-3,4-methylenedioxybenzyl)-1,3,4-oxadia-zolyl]carbonyl)-2-(S)-methylpropyl]-L-prolinamide. 42-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1- 44.4pyrimidinyl]-N-[1-(2-[5-tert-butyl-1,3,4-oxadiazol-yllcarbonyl-2-(R,S)-methylpropyl]acetamide 62-[5-Amino-6-Oxo-2-(4-fluorophenyl)-1,6-dihydro- 0.511-pyrimidinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)-methylpropyl]acetamide 82-[5-Amino-6-Oxo-2-phenyl-1,6-dihydro-1-pyri- 1.06midinyl]-N-[1-(2-[5-α,α-dimethyl-3,4-methylene-dioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)- methylpropyl]acetamide9 2-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N- 0.34[1-(2-[5(α,α-dimethyl-3,4-methylenedioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)-methyl- propyl]acetamide 10 2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimi- 1.53dinyl]-N-[1-(2-[5-(α,α-dimethyl-3,4-methylene-dioxybenzyl)-1,3,4-oxadiazolyl]carbonyl-2-(R,S)- methylpropyl]acetamide11  2-[6-Oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimi- 5.34dinyl]-N-[1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropy- flacetamide 12 2-[6-Oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl]-N- 1.83 [1-(2-[5-(α,α-dimethylbenzyl)-1,3,4-oxadiazolyl]carbonyl)-2-(R,S)-methylpropyl]acetamide

Example 15 Ex vivo inhibition of elastase

Sixty (60) minutes after the oral administration of an inhibitor with anappropriate vehicle, a blood sample (0.9 ml) is collected through theabdominal aorta by a syringe containing 0.1 ml of a 3.8% sodium citratesolution.

The blood sample is processed as follows: 60 μl of (final 0.1-1 mg/ml) asuspended solution of opsonized zymosan in Hank's buffer is added to thepreincubated whole blood (540 μl) for 5 minutes at 37 ° C., and theresulting mixture is incubated for 30 minutes at the same temperature.The reaction is terminated by immersing the test tube into ice water.The reaction mixture is then centrifuged at 3,000 rpm for 10 minutes at4° C. Twenty (20) μl of each of the resulting supernatant (the Sample)is measured for elastase activity.

The mixture consisting of the following components is incubated for 24hours at 37° C., and then optical density is measured at 405 nm:

0.2 M tris-HCI buffer (pH 8.0) 100 μl  2.5 M NaCl 40 μl Distilled water36 μl 50 mM solution of a substrate (*)  4 μl The Sample 20 μl*N-Methylsuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (SEQ ID NO: 1)

A test sample mixed with 1-methyl-2-pyrrolidone instead of the substrateis regarded as Substrate (−). A test sample mixed with saline instead ofthe Sample is regarded as Blank. The remaining elastase activity in theSample is calculated according to the following:

optical density of Substrate (+)−(optical density of Substrate(−)+optical density of Blank)

as a total production of p-nitroaniline over 24 hours based on astandard curve for the amount of p-nitroaniline.

An average activity is calculated based on the test sample of 5-6animals. An agent at 3, 10 or 30 mg/kg is orally given by a forcedadministration to a 24 hour fasted animal at 60 minutes before the bloodsampling. Optical density is measured by SPECTRA MAX 250 (MolecularDevices).

Some representative results are given in Table 3.

TABLE 3 Representative Results of Ex-Vivo Studies Percent Inhibition atIndicated Dosage Example 3 mg/kg 10 mg/kg 30 mg/kg 2  7%  9% 62% 3 44%69% 99%

1 1 4 PRT Artificial Sequence Sequence is a known commercially availablesubstrate for elastases. 1 Ala Ala Pro Val

What is claimed is:
 1. A compound of formula I

wherein R₁ is selected from tert-butyl andα,α-dimethyl-3,4-methylenedioxybenzyl; wherein the compound is selectedfrom the group consisting of: (i)methyloxycarbonyl-L-valyl-N-[1-(2-[5-α,α-dimethyl-3,4-methylenedioxybenzyl]-1,3,4-oxadiazolyl)carbonyl-2-(S)-methylpropyl]-L-prolinamide;and (ii)methyloxycarbonyl-L-valyl-N-[1-(2-[5-tert-butyl]-1,3,4-oxadiazolyl)carbonyl-2-(S)-methylpropyl]-L-prolinamide.